Process for manufacture of 9-amino-9-deoxyclavulanates

ABSTRACT

The compounds of the formula (II): ##STR1## and salts and esters thereof wherein R 1  is an inert organic group of up to 14 carbon atoms and R 2  is an inert organic group of up to 16 carbon atoms, the group NR 1  R 2  containing up to 22 carbon atoms, are antibacterial agents able to enhance the effectiveness of penicillins and cephalosporins against certain β-lactamase producing bacteria.

CROSS-REFERENCE

This is a division of Ser. No. 731,928 filed Oct. 13, 1976 now abandoned.

The present invention relates to antibacterial agents which also possess β-lactamase inhibitory activity, to their preparation and to pharmaceutical compositions containing them.

Belgian Pat. No. 827,926 discloses inter alia clavulanic acid, which is the compound of the formula (I): ##STR2## and its salts and esters. Clavulanic acid was shown to be a useful antibacterial agent which possesses β-lactamase inhibitory properties which allowed it to enhance the antibacterial effectiveness of penicillins and cephalosporins against many gram-negative and gram-positive bacteria. We have now discovered a further group of compounds that exhibit antibacterial and β-lactamase inhibitory activity but which have a different spectrum of activity than clavulanic acid and its salts.

The present invention provides compounds of the formula (II) ##STR3## and salts and esters thereof wherein R₁ is an inert organic group of up to 14 carbon atoms and R₂ is an inert organic group of up to 16 carbon atoms, the NR₁ R₂ group containing up to 22 carbon atoms.

When used herein the term `inert organic group` means any organic group that is itself stable and which does not contain any functional groupings which cause rupture of the β-lactam ring of the compound of the formula (II). Such groups do not include highly electron withdrawing groups situated in a manner which prevents the amine of the formula (III)

    HNR.sub.1 R.sub.2                                          (III)

from being sufficiently basic to form a salt of the carboxyl group of the compound of the formula (I) nor do they contain substituents that render the amine of the formula (III) unstable. Normally R₁ and R₂ are such that the conjugate acid of the amine of the formula H₂ NR₁ R₂ has a pKa of from 7.0 to 11.2 and preferably has a pKa of 8 to 10.

More suitably R₁ contains not more than 8 carbon atoms. More suitably R₂ contains not more than 14 carbon atoms. Most suitably the NR₁ R₂ group contains not more than 16 carbon atoms. Suitable values for R₁ include hydrocarbon groups (such as alkyl, alkenyl, phenylalkyl, phenylalkenyl or the like) optionally inertly substituted by halogen, OH, OR₃, O.CO₂ R₃, SR₃, COR₃ or the like whererin R₃ is a hydrocarbon group of up to 8 carbon atoms.

One group of particularly suitable values for R₁ is that of the sub-formula (a): ##STR4## wherein R₄ is a hydrogen atom or a C₁₋₄ alkyl group, R₅ is a hydrogen atom or a C₁₋₄ alkyl group and R₆ is an optionally substituted phenyl group.

More suitably R₄ is a hydrogen atom. More suitably R₅ is a hydrogen atom.

When used herein the term "optionally substituted phenyl Group" means a phenyl group or a phenyl group substituted by halogen, OH, OR₇, OCOR₇, CO₂ R₇ or COR₇ where R₇ is a hydrocarbon group of up to 7 carbon atoms and more suitably the term means a phenyl group or a phenyl group substituted by halogen, C₁₋₄ alkyl, C₁₋₄ alkoxyl or hydroxyl.

Particularly suitable groups of the sub-formula (a) include the benzyl, methoxybenzyl and chlorobenzyl groups, especially the benzyl group.

A further group of particularly suitable values for R₁ is that of the sub-formula (b): ##STR5## wherein each of R₈ to R₁₁ is independently a hydrogen atom or a C₁₋₄ alkyl group and R₁₂ is a C₁₋₄ alkyl group or an optionally substituted phenyl group or a hydrogen atom.

More suitably each of R₈ to R₁₁ is independently a hydrogen atom at or a methyl group. More suitably each of R₉ to R₁₁ is independently a hydrogen atom. More suitably R₁₂ is a hydrogen atom or a C₁₋₄ alkyl group.

Yet another particularly suitable group of values for R₁, is that of the sub-formula (c) ##STR6## wherein R₈ to R₁₂ are as defined in relation to sub-formula (b).

Particularly suitable groups R₂ include those of the sub-formula (a), (b) and (c) previously described as suitable for R₁. The group R₂ need not have the same value as the group R₁ but it is sometimes convenient for it to be so.

Other particularly suitable groups R₂ include those of the sub-formula (d):

    --CHR.sub.13 R.sub.14                                      (d)

wherein R₁₃ is a hydrogen atom or a C₁₋₄ alkyl group and R₁₄ is an alkyl group of 1-14 carbon atoms optionally substituted by OH, OR₁₆, OCOR₁₆ or COR₁₆ where R₁₆ is a hydrocarbon group of up to 8 carbon atoms.

When R₁₃ is a hydrogen atom it is preferred that R₁₄ is not a methyl group.

Particularly suitable groups of the sub-formulae (d) include the isopropyl, 2-hydroxyethyl, 2-hydroxypropyl, ethyl, 2-acetoxyethyl, 2-methoxyethyl and the like group.

Suitable esters of the formula include those of the formulae (IV) and (V): ##STR7## and acid addition salts thereof wherein R₁ and R₂ are as defined in relation to formula (II) and A¹ is an alkyl group of 1-8 carbon atoms optionally substituted by halogen or a group of the formula OA⁴, OCOA⁴, SA⁴, SO₂ A⁴ wherein A⁴ is a hydrocarbon group of up to 6 carbon atoms; A² is a hydrogen atom, an alkyl group of up to 4 carbon atoms or a phenyl group optionally substituted by halogen or by a group A⁵ or OA⁵ where A⁵ is an alkyl group of up to 6 carbon atoms; and A³ is a phenyl group optionally substituted by halogen or by a group A⁵ or OA⁵ where A⁵ is an alkyl group of up to 6 carbon atoms.

Suitable acid addition salts include those formed with pharmaceutically acceptable acids which are known to be suitable for forming salts with esters of penicillins or cephalosporins which contain a basic group.

A further suitable group of esters are those which are readilly in-vivo hydrolysable which include, but are not limited to, those of the formulae (V) and (V): ##STR8## wherein A₁ is a hydrogen atom, alkyl, aryl or aralkyl group; A₂ is a hydrogen atom or methyl group; A₃ is an alkyl, aryl or aralkyl group; X is oxygen or sulphur; Y is oxygen or sulphur and Z is a divalent organic group. Esters of the formulae (V') and (VI') which fairly readily release the clavulanic acid into the blood stream after administration include those wherein A₁ is a hydrogen atom, A₂ is a hydrogen atom or a methyl group and A₃ is a methyl, ethyl, propyl, butyl, benzyl, or phenyl group and those wherein X is oxygen, Y is oxygen and Z is --CH₂ CH₂ --, --CH:CH--, ##STR9##

When used in conjunction with the preceeding formula the term `alkyl` includes alkyl of up to six carbon atoms; the term `aryl` includes phenyl, naphthyl or phenyl substituted by an inert substituent such as a fluorine or chlorine atom or a methyl or methoxyl group or the like; when used herein the term `aralkyl` means an alkyl group substituted by an aryl group.

Particularly suitable esters of the formulae (V') and (VI') include those of the formulae (VII') and (VIII'): ##STR10## A₄ is a hydrogen atom or a methyl group, A₅ is a methyl, t-butyl or phenyl group and A₆ is a hydrogen atom or a methoxyl group.

Many esters of clavulanic acid differ from analagous esters of penicillins or cephalosporins in that they show an enhanced tendency to hydrolyse to clavulanic acid under mild conditions. Thus, for example, simple alkyl esters such as the methyl ester slowly hydrolyse to clavulanic acid in water buffered to pH7. Esters which undergo some hydrolysis under mild conditions are included within the formula (IX'): ##STR11## wherein R¹ is a hydrocarbon group of 1-9 carbon atoms optionally substituted by halogen, lower alkoxy, hydroxyl or optionally salted basic groups of the formula NR² R³ wherein R² is a hydrogen atom or a lower alkyl group, R³ is a hydrogen atom or a lower alkyl group or is attached to R² so that NR² R³ is a 5- or 6-membered ring.

When used with reference to formula (IX') the term `lower` means that the group contains 1-4 carbon atoms.

Suitably groups R¹ include alkyl and aralkyl groups optionally substituted by halogen, methoxyl, hydroxyl or salted NR² R³ groups wherein R² is a methyl or ethyl group and R³ is a methyl or ethyl group or is joined to R² so that NR² R³ is a pyrrolidine, piperidine or morpholine group.

Most suitably alkyl groups R¹ are straight chain groups of up to 6 carbon atoms optionally substituted by one methoxyl, hydroxyl, salted NR² R³ group or one chlorine, bromine or iodine atom or by a CCl₃ or CF₃ group. Suitable in-vivo hydrolysable esters include phthalidyl, acetoxymethyl, pivaloyloxymethyl, αacetoxyethyl, ethoxycarbonyloxymethyl, αethoxycarbonyloxyethyl and the like esters.

When non-esterified, the compounds of this invention are normally salted. Such salts are preferably zwitterionic salts of the formula (VI). ##STR12## Other salts of the compounds of formula (II) include those with pharmaceutically acceptable salting ions such as the sodium, potassium, calcium, magnesium, aluminium, ammonium, and substituted ammonium ions and also those with pharmaceutically acceptable acids. These other salts are not a preferred feature of the invention. From the preceeding statements it will be realised that certain particularly suitable compounds of this invention are of the formulae (VII)-(IX) ##STR13## and in-vivo hydrolysable esters thereof wherein R₆ and R₁₂ are as described in relation to sub-formulae (a), (b) and (c) and R₁₃ is a CH₂ R₆, CH₂ CH: CHR₁₂, CH₂ --CH₂ --CH₂ --R₁₂ or CHR₁₃ R₁₄ group where R₁₃ and R₁₄ are defined as in relation to sub-formula (d).

Compounds within formulae (VII) can be particularly effective in inhibiting β-lactamases produced by gram-positive bacteria. Compounds within formulae (VII) and (IX) are envisaged as useful for their broad spectrum of β-lactamase inhibition.

The present invention provides a process for the preparation of the compounds of the formula (II) and salts and esters thereof which process comprises the reaction of an amine of the formula (III)

    HNR.sub.1 R.sub.2                                          (III)

wherein R₁ and R₂ are as defined in relation to formula (II), and a compound of the formula (X). ##STR14## where CO₂ A is an ester group and thereafter if desired converting the thus formed compound into the corresponding carboxylic acid or a salt.

The diene of the formula (X) may be formed before the reaction with the amine of the formula (III) or it may be prepared in situ. Suitable methods of preparing the diene in situ include the displacement of sulphate or carboxylate moiety from a compound of the formulae (XI) or (XII) ##STR15## wherein A is as defined in relation to formula (X), Q¹, Q² and Q³ are groups such that NQ¹, Q², Q³ is a tertiary amine and Q⁴ is an organic group.

Suitably the reaction of the amine of the formula (III) with the compound of the formula (X), (XI) or (XII) will take place in an aprotic solvent such as acetonitrile, dimethylformamide or other similar solvent at a non-extreme temperature, for example--10° to 50° C., more usually--5° to 25° C. and conveniently within the range 0° to 20°.

Suitably one or more of Q¹, Q² and Q³ is a C₁₋₆ alkyl group such as a methyl or ethyl group.

Suitably Q⁴ is a C₁₋₆ alkyl, benzyl, dichloromethyl or like group.

It is frequently advantageous to use the preformed diene ester of the formula (X) rather than to generate it in situ.

When a compound of the formula (XI) or (XII) is used in the process of this invention a certain degree of direct displacement of the leaving group by the amine may take place but it is believed that in general most and possibly effectively all of the desired compound is produced via the diene.

The present invention also provides a process for the preparation of acids of the formula (II) and the carboxylate salts thereof which process comprises the de-esterification of a corresponding ester of the compound of the formula (II) and if desired simultaneously or subsequently salting the carboxyl group.

De-esterification may be brought about by conventional mild methods such as hydrogenation or mild basic hydrolysis.

Suitable hydrogenolysable esters of the compound of the formula (II) include benzyl and like esters. Such esters may be cleaved by hydrogenation using a low or medium pressure of hydrogen, for example about 1 atmosphere, at an approximately ambient temperature, for example about 12°-20° C., in a conventional inert solvent, for example ethanol.

If a base is present a basic salt maybe formed. Normally no base is present and the zwitterionic salt results.

Suitable base hydrolysable esters include acetoxymethyl, phthalidyl and the like ester which undergo hydrolysis when maintained in an aqueous medium at a pH of 7 to 8.5. Such reactions can occur rapidly, for example in 10-60 minutes. Most suitably such reactions take place in a solvent which is water or water together with an organic solvent such as tetrahydrofuran. The reaction usually occurs sufficiently rapidly at 5°-20° C. The pH may be maintained at the correct level by the careful addition of base. This is generally a less suitable method of cleaving the ester than hydrogenolysis.

Compounds of the formula (II) where both or one of R₁ and R₂ is a group of the sub-formula (c) as herein before described may be prepared by the reduction of a corresponding compound containing a corresponding group of the sub-formula (b). Such reactions are normally effected by hydrogenation in the presence of a transition metal catalyst in conventional manner.

The present invention also provides pharmaceutical compositions which comprise a compound of this invention and a pharmaceutically acceptable carrier.

The compositions of the invention include those in a form adapted for oral, topical or parenteral use and may be used for the treatment of infection in mammals including humans.

Suitable forms of the compositions of this invention include tablets, capsules, creams, syrups, suspensions, solutions, reconstitutable powders and sterile forms suitable for injection or infusion. Such compositions may contain conventional pharmaceutically acceptable materials such as diluents, binders, colours, flavours, preservatives, disintegration and the like in accordance with conventional pharmaceutical practice in the manner well understood by those skilled in the art of formulating antibiotics.

Injectable or infusable compositions of salts of a compound of the formula (II) are particularly suitable as high tissue levels of a compound of the formula (II) can occur after administration by injection or infusion. Thus, one preferred composition aspect of this invention comprises a salt of a compound of the formula (II) in sterile form.

Unit dose compositions comprising a compound of the formula (II) or a salt or ester thereof adapted for oral administration form a further preferred composition aspect of this invention.

The compound of the formula (II) or its salt or ester may be present in the composition as sole therapeutic agent or it may be present together with other therapeutic agents such as a β-lactam antibiotic. Suitable β-lactam antibiotics for inclusion in the compositions of this invention include benzylpenicillin, phenoxymethylpenicillin, carbenicillin, azidocillin, propicillin, ampicillin, amoxycillin, epicillin, ticarcillin, cyclacillin, cefatriazine, pirbenicillin, α-sulphonyloxybenzylpenicillin, cephaloridine, cephalothin, cefazolin, cephalexin, cefoxitin, cephacetrile, cephamandole nafate, cephapirin, cephradine, 4-hydroxycephalexin, cefaparole, cephaloglycine, and other well known pencillins and cephalosporins or pro-drugs therefore such as hetacillin, metampicillin, 4-acetoxyampicillin, the acetoxymethyl, ethoxycarbonyloxymethyl, pivaloyloxymethyl or phthalidyl esters of benzylpenicillin, ampicillin, amoxycillin or cephaloglycine or the phenyl, tolyl or indanyl α-esters or carbenicillin or ticarcillin or the like. Such compounds are frequently used in the form of a salt or hydrate.

Naturally if the penicillin or cephalosporin present in the composition is not suitable for oral administration then the composition will be adapted for parenteral administration.

When present in a pharmaceutical composition together with a β-lactam antibiotic, the ratio of a compound of the formula (II) or its salt or ester present to β-lactam antibiotic present may vary over a wide range of ratios, for example 10:1 to 1:3 and advantageously may be from 5:1 to 1:2, for example, 3:1 to 1:1.

The total quantity of antibacterial agents present in any unit dosage form will normally be between 50 and 1500 mg and will usually be between 100 and 1000 mg.

Compositions of this invention may be used for the treatment of infections on inter alia, the respiratory tract, the urinary tract and soft tissues and mastitis in cattle.

Normally between 50 and 3000 mg of the compounds of the invention will be administered each day of treatment but more usually between 100 and 1000 mg of the compounds of the invention will be administered per day, for example as 1-6 doses, more usually 2-4 doses.

The penicillin or cephalosporin in synergistic compositions of this invention will normally be present by up to or at approximately the amount at which it is conventionally used.

Particularly favoured compositions of this invention will contain from 150-1000 mg of amoxycillin, ampicillin or a pro-drug therefore and from 50-500 mg of a compound of the formula (II) or a salt or in-vivo hydrolysable ester thereof and more suitably from 200-500 mg of amoxycillin, ampicillin or a pro-drug therefore and from 50-250 mg of a compound of the formula (II) or a salt or in-vivo hydrolysable ester thereof.

The materials present in such compositions may be hydrated if required, for example ampicillin trihydrate or amoxycillin trihydrate may be employed. The weights of the antibiotics in such compositions are expressed on the basis of antibiotic theoretically available from the composition and not on the basis of the weight of pro-drug.

EXAMPLE 1 Benzyl 3-(2-dibenzylaminoethylidene)-7-oxo-1-azabicylo[3.2.0]heptane-2-carboxylate (e2) ##STR16##

Crude benzyl clavulanyl sulphate trimethylammonium salt (e1) (500 mg) in freshly distilled dimethylformamide (5 ml) was treated with a solution of dibenzylamine (450 mg) in dimethylformamide (2 ml) dropwise over 15 minutes. The reaction was stirred at room temperature for 2 hours and diluted with ethyl acetate/petroleum ether (b.p. 60-80) 1/1 (50 ml). The solution was chromatographed directly on silica gel to yield as the first eluted product benzyl dibenzylaminodeoxy clavulanate (e2) as a light yellow oil (84 mg.) Rechromatography afforded pure (e2) as a colourless oil (38 mg.)

The preparation of (e1) is described in German Patent Application No. 2616087 and United States Patent application Ser. No. 675,273

N.m.r. (CDCl₃): 3.00 (1H, d, J=17 Hz, 6β-CH); 3.20 (2H, d,J=8 Hz), CH₂ N); 3.52 (1H, dd, J=17 Hz, 6α-CH); 3.57 [4H, s, N(CH₂ Ph)₂ ]; 4.84 (1H, br.t., J=8 Hz, olefinic CH); 5.13 (iH, br.s., 3-CH); 5.25 (2H, s, CO₂ CH₂ Ph); 5.69 (1H, d, J=2.5 Hz, 5-CH); 7.41δ(15H, s, aromatic protons).

When the compound prepared by the method of Example 1 was tested against certain enzymes by the β-lactamase inhibition assay described in Belgian Patent No. 827926 the following ^(I) 50 values were obtained:

                         Approximate                                               Enzyme               I.sub.50 (in mg/ml)                                       ______________________________________                                         E. coli JT4          0.02                                                      E. coli JT10         0.24                                                      Klebsiella aerogenes AE70                                                                           0.40                                                      Staphylococcus aureus (Russell)                                                                     0.06                                                      Pseudomonas aeruginosa                                                                              0.14                                                      Citrobacter mantio   0.15                                                      ______________________________________                                    

EXAMPLE 2 Methyl 3-(2-dibenzylaminoethylidene)-7-oxo-4-1-azabicyclo[3,2,0]2-carboxylate ##STR17## The compound (e2) (100 mg) in tetrahydrofuran (5 ml) was hydrogenated at ambient temperature and pressure over 10% Pd/C (50 mg) for one hour. The solution was filtered through keirselguhr, the solvent evaporated, the residue dissolved in ethylacetate (10 ml) and extracted with water (4×10 ml). The water was evaporated to yield a gum which was dissolved in methanol (5 ml) and treated with excess/diazomethane at 0° C. Evaporation of the solvent and chromatography yield the desired compound (methyl dibenzylamino deoxyclavulanate (e3) as a colourless gum.

i.r. (CHCl₃) 1800, 1755, 1700 cm⁻¹

n.m.r. (CDCl₃) 2.94 (1H,d,J=17 Hz, 6β-CH); 3.17 (2H, d, J=7 Hz, ═CH-CH₂); 3.41 (1H, dd, J=17 Hz, J¹ =2.5 Hz, 6α-CH); 3.53 (4H,s, N[CH₂ C₆ H₅ ]₂); 3.74 (3H, s, CO₂ CH); 4.78 (1H,brit, J=7 Hz, ═CH-CH₂) 5.01 (1H, br. s, 3-CH); 5.60 (1H, d, J=2.5 Hz, 5-CH) 7.30 δ(10H,s N[CH₂ C₆ H₅ ]₂ [α]²⁵ =0.4° (C=0.81; MeOH).

EXAMPLE 3 Benzyl N-benzyl-N-2-hydroxyethylamino-deoxyclavulanate ##STR18## Clavudiene benzyl ester (0.5 g) in acetonitrile (10 ml) was cooled to 0° C., N-benzyl-2-hydroxyethylamine (0.36 g) was added and the reaction mixture stirred for 21/2 hours. Ethyl acetate (100 ml) was added and the mixture evaporated to low volume. The residue was subjected to column chromatography using ethyl acetate as eluent. The product, was isolated in low yield had an ir spectrum (liquid film) as follows: 3400 (broad, --OH), 1800 (β-lactam C═O), 1740 (ester C═O), 1700 (C═C), 695 cm⁻¹ (aromatic protons). The n.m.r. spectrum was consistent with the desired product (e5). EXAMPLE 4 Benzyl N-benzyl-N-isopropylamino deoxyclavulanate ##STR19## The ester(e4)(0.5 g) in acetronitrile (10 ml) was cooled in ice-water. N-iso propylbenzylamine (0.39 g, 1.3 moles) was added with stirring. The reaction mixture was allowed to warm to room temperature and stirred for 3 hours. Ethyl acetate (100 ml) was added, and the solution evaporated to low bulk in vacuo. The residue was subjected to column chromatography on silica gel using cyclohexane and ethyl acetate as eluents. The product (e6) was eluted after the unreacted diene. It had Ir spectrum (liquid film) as follows: 1803, (β-lactam C═O) 1845 (ester C═O) 1700 (C═C) 695 cm⁻¹ (aromatic protons). The n.m.r. spectrum was consistent with the desired product EXAMPLE 5 Benzyl dibenzylaminodeoxyclavulanate ##STR20## Benzyl dichloroacetylclavulanate (e7) (0.8 g) was dissolved in dry dimethylformamide and cooled to 0° C., treated with dibenzylamine (768 μl; 0.004 mol) in dry dimethylformamide (4 ml) over 15 minutes, the temperature being maintained at 0° C. The resulting yellow solution was stirred at 0° for 21/2 hours and a room temperature for 4 hours. Ethylacetate was added (100 ml) and the solution washed with water (3×25 ml), dried and evaporated. The product was purified by fast gradient elution on silica gel using ethyl acetate/cyclohexane as the eluting solvent (Yield 0.33 g).

n.m.r. (CDCl₃)2.92 (d, IH,J 17 Hz, 6β-H) 3.12 (2H, d, J 8 Hz, ═CH CH₂ N) 3.38 (IH,dd,J=17 Hz, J¹ 2.5 Hz, 6α-H) 3.46 (4H,s,N(CH₂ Ph)₂ 4.72 (IH, dt, J 8 Hz,=CH CH₂)5.01 (IH,bs, CH CO₂ B_(z)) 5.12 (2H, s CO₂ CH₂ Ph) 5.53 (IH,d, J 2.5 Hz, 5-CH) 7.22 (15H,S, aromatic-H)

EXAMPLE 6 Benzyl dibenzylaminodeoxyclavulanate ##STR21## The diene (e4) (271 mg) in dry acetonitrile (4 ml) at 0° C. was treated with dibenzylamine (197 mg) in dry acetonitrite (2 ml) over 5 minutes. The reaction mixture was stirred at 0° C. for 2 hours and at room temperature for 2 hours. The solvent was removed by evaporation and the residue dissolved in ethyl acetate, washed with water, dried evaporated and fractionated on silica-gel to yield the desired product (e2) which was purified by chromatography. EXAMPLE 7 Benzyl N-benzyl-9-(dl-2-hydroxypropylamino-9-deoxyclavulanate ##STR22## Benzyl dichloroacetylclavulanate (e7) (0.8 g) in dry dimethylformamide (20 ml) was cooled to 0° C. and a solution of dl-1-benzylamino-2-propanol (0.65 g) in dry dimethylformamide was added slowly. Stirring was continued for 4 hours at 0° C.

A more polar component was formed (thin layer chromatograph) and worked up as described in example 5 and chromatographed to give the desired product (e8) (0.16 g)

i.r. (film)3450, 1808, 1750, 1700 cm⁻¹

n.m.r. (CDCl₃) 0.9 ##STR23## 2.97 (1H, dd, J 17.5 Hz, J¹ 1.5 Hz 6β-H) 3.17 (2H, d, J 7 HZ, ═CH.CH₂) 3.56 (1H, dd, J 17.5 HZ, J¹ 2.5 HZ, 6αH) 3.48 (2H, s N CH₂ Ph) 4.75 (1H, bt, J 7 Hz, ═CH CH₂) 5.1 (1H, s, C-3) 5.18 (2H,s, CO₂ CHph) 5.63 (1H, d,J 2.5 Hz 5-H) 7.21, 7.3 (10H, s, aromatic-H)

EXAMPLE 8 Benzyl diallylaminodeoxyclavulanate ##STR24## Benzyl dichloroacetylcalvulanate (e7) (0.4 g) was dissolved in dry dimethylformamide (7 mls) and cooled to 0° C. A solution of diallylamine (246 μl; 0.002 mol) in dry dimethylformamide (2 ml) was added the temperature was maintained at 0° C. The solution was stirred for 2 hours at 0° and at 15° for 15 minutes. Ethyl acetate (75 ml) was added and the solution washed with water (2×35 ml) dried and evaporated. The more polar product was isolated by column chromatography on silica gel and was obtained as a yellow oil (0.18 g);

i.r.(film) 1804, 1755, 1695, 1640 cm⁻¹.

n.m.r. (CDCl₃), 2.96 (IH,d, J 17 Hz, 6β-H) 2.97 (4H, d, J 6 Hz, N(CH₂ CH═CH₂) 3.13 (2H, d, J 7 Hz, ═CH CH₂ N<), 3.41 (IH,dd,J' 2.5 Hz 6α-H) 4.68 (1H,dt, J 7 Hz, ═CH CH₂)4.97-5.13 (5H, m,3-CH and N[CH₂ CH═CH₂ ]₂), 5.13 (2H, s superimposed on broad m, CO₂ CH₂ Ph), 5.62 (1H, d, J 2.5 Hz, 5-H), 5.75 (2H, m, N[CH₂ CH═CH₂ ]), 7.3 (5H, s, CH₂ Ph).

EXAMPLE 9 p-Methoxybenzyl diallylaminodeoxyclavulanate ##STR25## The sulphate (e10) (0.46 g) was dissolved in dry dimethylformamide (7 ml) and cooled to 0° C., diallylamine (0.175 g) in dry dimethylformamide (2 ml) was added slowly dropwise. The reaction mixture was stirred at 0° for 2 hours and worked up as in Example 5. Column chromatograph gave the product (e11) as a yellow oil. EXAMPLE 10 9-(NN-dibenzylamino)-9-deoxyclavulanic acid ##STR26##

A solution of the benzyl ester (e2) (330 mg) in ethanol (20 ml) was hydrogenolysed at ambient temperature and pressure using 10% Pd/C (110 mg). The reaction was complete after 15 minutes.

The catalyst was filtered off and the filtrate evaporated, the crude product was dissolved in ethyl acetate (20 ml) and extracted with water (5×5 ml). The water was removed in vacuo to give the desired product (e12) as a pale yellow gum (yield 41%).

i.r. 3400 (b), 1800, 1700 (W), 1620 (b) cm⁻¹

EXAMPLE 11 Benzyl N-benzyl-N-norbornylaminodeoxyclavulanate ##STR27##

Clavudiene benzyl ester (0.5 g) in acetonitrile (10 ml) was treated at -15° with 2 (N-benzyl) norbornylamine (1.3 moles, 0.48 g). Allowed to warm to room temperature during about 2 hours, stood 1 hour, added ethyl acetate (100 ml), evaporated to small volume. Residue subjected to column chromatography on silica gel using gradient elution with cyclohexane and ethyl acetate. The solvents were removed under reduced pressure to yield the desired product (e13) as a pale yellow oil.

I.r. spectrum as follows: 1802 cm⁻¹ (β-lactam C═O).

N.m.r. 0.9-3.7 (unassignable multiple peaks), 4.65 (1H,t, CH═), 4.97 (2H, C₆ H₅ CH₂), 5.1 (1H,s, 3-CH)

EXAMPLE 12 9-(N,N-dipropylamino)-9-deoxyclavulanio acid ##STR28##

Benzyl 9-(N, N-diallylamino)-9-deoxyclavulanate (e9) was dissolved in ethanol (10 ml) and hydrogenolysed at room temperature and pressure using 10% Pd/C catalyst (70 mg). After filtration through kieselguhr the solvent was evaporated and the residue dissolved in ethyl acetate (25 ml) and the product extracted with water (3×10 ml). Evaporation in vacuo gave the zwitterion (e14) as a yellow gum in 85% yield.

(i.r. (film) 3400 (b), 2500 (b), 1790, 1695, 1625 cm⁻¹

n.m.r. (D₂ O) 0.83 (6H, t, J 7 Hz N (CH₂ CH₂ CH₃)₂) 1.6 (4H, m, N(CH₂ CH₃)₂) 2.9 (5H, m, 6β-H, N(CH₂ CH₂ CH₃)₂) 3.25 (IH, dd, J 18 Hz, J¹ 3 Hz, 6 -H) 3.75 (2H, d, J 8 Hz, ═CH.CH₂) 4.66 (IH, t,obscured by HOD peak, ═CH CH₂) 4.95 IH, s,-5.72 (IH, d, J 3 Hz, 5-H).

EXAMPLE 13 9-[DL-N-Benzyl-N-(2-hydroxypropyl)amino]-9-deoxyclavulanic acid ##STR29##

The zwitterion was prepared by hydrogenolysis of benzyl-9-[DL-N-benzyl-N-(2-hydroxypropyl)amino]-9-deoxyclavulanate (e 8) (90 mg) using 10% pd/C catalyst (33 mg) in ethanol (10 ml) at ambient temperature and pressure. The reaction was complete in 30 minutes. The catalyst was removed by filtration through kieselguhr and the filtrate was evaporated. The residual gum was taken up in ethyl acetate (20 ml) and extracted with water (4×10 ml). The aqueous extracts were combined and evaporated in vacuo to give the desired product (e15) as a pale yellow gum in 52% yield.

i.r. (film) 3400 (b) 1790, 1695, 1620 cm³¹ 1.

The Field Desorption mass spectrum showed a peak at m/e 347 corresponding to (M+1) and a peak at m/e 165 (PhCH₂ N⁺ HCH₂ CHOHCH₃).

EXAMPLE 14 Activity

a. The compounds of this invention do not have a high level of acute toxicity, for example the compounds of Examples 8 and 12 have LD₅₀ values of greater than 500 mg/kg in mice when administered by the sub-cutaneous or oral routes.

b. The effectiveness of the compounds of this invention as synergists can be demonstrated by conventional MIC tests in which ampicillin alone, ampicillin and synergist and synergist alone are compared. The following test results were obtained using a strain of Staphylococcus aureus Russell which is neither inhibited by 250 μg/ml of ampicillin and nor inhibited by 5 μg/ml of synergist administered separately.

    ______________________________________                                                            MIC of ampicillin                                           Compound of Example                                                                               (μg/ml) in presence                                      No.                of 1 μg/ml of synergist                                  ______________________________________                                         2                  3.12                                                        1                  7.8                                                         8                  <0.09                                                       10                 0.156                                                       ______________________________________                                    

PREPARATION 1 General Procedure for Preparation of Sulphonate

The following process for the preparation of benzyl clavulanate --O-- sulphonate trimethylamine salt (e1) may be adapted for the preparation of analogous esters by substituting the appropriate ester of clavulanic acid for (e1).

Benzyl clavulanate (57.8 mg) in dry dimethylformamide (0.8 ml) was treated with the trimethylamine-sulphur trioxide complex (55.6 mg) and left at ambient temperature for 18 hours. The solvent was removed in vacuo and the residue extracted with chloroform. The chloroform solution was evaporated and the residue was extracted several times with diethyl ether to remove benzyl clavulanate. The ether insoluble oil was shown to be the trimethylamine salt of benzyl clavulanate --O-- sulphonate (e1) which was produced in 60% yield. (In the n.m.r. the --CH₂ OSO₃ ⁻ proton appeared at a doublet at about 4.71δ as compared to about 4.24δ in the starting material).

PREPARATION 2 General Procedure for Preparation of Diene Esters

The following process for the preparation of the benzyl ester of clavuladiene may be adapted to the preparation of other corresponding esters by replacing the benzyl clavulanate starting material by the corresponding ester of clavulanic acid.

Benzylclavulanate (0.2 g) was added to dry dimethylsulphoxide (6 ml) and dry benzene (3 ml) containing dicyclohexylcarbodiimide (0.43 g.). Anhydrous orthophosphoric acid (0.069 g) in dimethyl sulphoxide (2 ml) was added and the mixture stirred at room temperature for 4 hours. Thin layer chromatography showed a faster moving spot which gave a blue fluorescence at 366 n.m. The dicyclohexylurea was filtered off and benzene added to the filtrate, the organic phase was washed with water dried and evaporated. Fractionation on silica gel gave the product as a colourless oil in 71% yield. The diene was stored as a solution in acetone containing hydroquinone (0.01%) as a stabiliser.

I.r. (film): 1810, 1700, 1628, 1565 cm⁻¹

EXAMPLE 15 Methyl 2-(N-benzyl) norbonylaminodeoxyclavulanate ##STR30## Sodium clavulanate tetrahydrate (e16) (2.9 g) in dimethylformamide (25 ml) was treated with iodomethane (25 ml) at room temperature over 1 hour. Acetontrile (10 ml) was added and removed by evaporation under reduced pressure (to remove excess methyliodide). The residual dimethylformamide solution was cooled in ice, and phthalic anhydride (1.5 g) and triethylamine (5 ml) added. After 1 hour at below 5° C., acetonitrile (2×25 ml portions) was added and evaporated under reduced pressure each time. 2-N-benzylnorbornylamine (4 g) was added and the reaction allowed to stir at 5° C. overnight. Most of the dimethylformamide was evaporated in vacuo. The residue was dissolved in 1:1 ethyl acetate-cyclohexane (100 ml), treated with silica gel (15 g) (chromatography grade), filtered off, evaporated to small bulk, and the residue subjected to gradient chromatography on silica gel using ethyl acetate and cyclohexane as eluents. The least polar β-lactam containing material was isolated by evaporation of the solvents from fractions containing it, to yield methyl 2-(N-benzyl)norbornylaminodeoxyclavulanate (e17) as a yellow oil (50 mg). It had I.r. 1800 (β-lactam) 1750 (ester C═O) 1695 cm⁻¹ (C═C). EXAMPLE 16 Benzyl N-benzyl-N-ethylaminodeoxyclavulanate ##STR31## Clavudiene benzyl ester (0.44 g) in acetonitrile (8 ml) was cooled to -10° C. and treated with N-benzylethylamine (0.27 g). The reaction mixture was maintained at -5° to 0° C. for 11/2 hours. Ethyl acetate (70 ml) was added and the mixture evaporated to small volume. The residue was subjected to column chromatography on silica gel using a gradient elution, starting with 7:1 cyclohexane/ethyl acetate increasing quickly to 2:1.

The material was collected in fractions just after the starting material. The solvents were evaporated to yield 45 mg of the title compound (e18) as a pale yellow oil. I.r. 1803 (β-lactam) 1750 (ester) 1700 (C═C) cm⁻¹. 

What is claimed is:
 1. A process for the production of a compound of the formula (II): ##STR32## or an ester thereof of the formula (IV) or (V): ##STR33## wherein A¹ is alkyl of 1-8 carbon atoms unsubstituted or substituted by halogen or a group of the formula OA⁴, OCOA⁴, SA⁴ or SO₂ A⁴ wherein A⁴ is a hydrocarbon of up to 6 carbon atoms; A² is hydrogen, alkyl of up to 4 carbon atoms or phenyl unsubstituted or substituted by halogen or by A⁵ of OA⁵ wherein A⁵ is alkyl of up to 6 carbon atoms; and A³ is phenyl unsubstituted or substituted by halogen or by A⁵ of OA⁵ wherein A⁵ is as above defined; and wherein R₁ and R₂ are each independently a group of the sub-formula (a) (b) or (c):(a) --CR₄ R₅ R₆ (b) --CR₈ R₉ --C(R₁₀)═CR₁₁ R₁₂ (c) --CR₈ R₉ --CH(R₁₀)--CHR₁₁ R₁₂ wherein R₄ is hydrogen; R₅ is hydrogen or alkyl of 1-4 carbon atoms; R₆ is phenyl unsubstituted or substituted by a substituent selected from the group consisting of halogen, OH, OR₇, OCOR₇, and COR₇, wherein R₇ is a hydrocarbon of up to 7 carbon atoms; R₈ is hydrogen or methyl; R₉ is hydrogen or methyl; R₁₀ is hydrogen or methyl; R₁₁ is hydrogen or methyl; or R₁₂ is hydrogen, alkyl of 1 to 4 carbon atoms or phenyl unsubstituted or substituted by a substituent selected from a group consisting of halogen, OH, OR₇, OCOR₇, CO₂ R₇ and COR₇ wherein R₇ is a hydrocarbon of up to 7 carbon atoms which comprises reacting an amine of the formula HN₁ R₂ (III) wherein R₁ and R₂ are as above defined, with a compound of the formula (X): ##STR34## wherein CO₂ A is an ester as above defined and in the case of the acid, hydrogenating the ester produced to the corresponding acid.
 2. A process according to claim 1 wherein compound (II) is of the formula: ##STR35## wherein R₄ is hydrogen, R₅ is hydrogen or alkyl of 1 to 4 carbon atoms and R₆ is phenyl unsubstituted or substituted by a substituent selected from the group consisting of halogen, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms or hydroxyl.
 3. A process according to claim 2 wherein R₅ is hydrogen.
 4. A process according to claim 2 wherein CR₄ R₅ R₆ is benzyl, methoxybenzyl or chlorobenzyl.
 5. A process according to claim 1 wherein compound (II) is of the formula: ##STR36## wherein each of R₈ to R₁₁ is independently hydrogen or methyl and R₁₂ is hydrogen, alkyl of 1 to 4 carbon atoms or phenyl unsubstituted or substituted by a substituent selected from the group consisting of halogen, alkyl of 1 to 4 carbon atoms, alkoxyl of 1 to 4 carbon atoms or hydroxyl.
 6. A process according to claim 5 wherein each of R₉ to R₁₁ is hydrogen.
 7. A process according to claim 6 wherein R₁₂ is hydrogen or alkyl of 1 to 4 carbon atoms.
 8. A process according to claim 1 wherein compound (II) is of the formula: ##STR37## wherein each of R₈ to R₁₁ is independently hydrogen or methyl and R₁₂ is hydrogen, alkyl of 1 to 4 carbon atoms or phenyl unsubstituted or substituted by a substituent selected from the group consisting of halogen, alkyl of 1 to 4 carbon atoms, alkoxyl of 1 to 4 carbon atoms or hydroxyl.
 9. A process according to claim 8 wherein each of R₉ to R₁₁ is hydrogen.
 10. A process according to claim 9 wherein R₁₂ is hydrogen or alkyl of 1 to 4 carbon atoms.
 11. A process according to claim 1 wherein compound (II) is of the formulae (VII) to (IX): ##STR38## wherein R₆ is phenyl or phenyl mono-substituted by halogen, alkyl of 1 to 4 carbon atoms, alkoxyl of 1 to 4 carbon atoms or hydroxyl, R₁₆ is CH₂ R₆, CH₂ CH═CHR₁₂, CH₂ CH₂ CH₂ R₁₂ or CHR₁₃ R₁₄ wherein R₆ is as above defined, R₁₂ is alkyl of 1 to 4 carbon atoms, phenyl unsubstituted or mono-substituted by halogen, OH, alkyl, alkoxyl of 1 to 4 carbon atoms, or hydrogen, R₁₃ is hydrogen or alkyl of 1 to 4 carbon atoms, R₁₄ is alkyl of up to 14 carbon atoms unsubstituted or mono-substituted by OH, OR₁₅ or COR₁₅ wherein R₁₅ is alkyl, alkenyl, phenylalkyl or phenylalkenyl of up to 8 carbon atoms.
 12. A process according to claim 1 wherein the ester is the phthalidyl, acetoxymethyl, pivaloyloxymethyl, α-acetoxyethyl, ethoxycarbonyloxymethyl or α-ethoxycarbonyloxyethyl ester.
 13. A process according to claim 1 for production of the benzyl ester of the compound of the formula (II) wherein A is benzyl.
 14. A process according to claim 1 wherein the reaction takes place in an aprotic solvent at a temperature of from -10° C. to +50° C.
 15. A process according to claim 14 wherein the solvent is acetonitrile or dimethylformamide.
 16. A process according to claim 14 wherein the temperature is from -5° C. to +25° C.
 17. A process according to claim 1 wherein the temperature is from 0° C. to 20° C. 